Hypertrophic cardiomyopathy (HCM) affects 1 in 500 general population with variable clinical course, and underlying mechanism of hypertrophy or basis for clinical heterogeneity. There have been over 300 mutations in the ?-cardiac myosin heavy chain (MyHC) gene that cause HCM, but the underlying molecular effects on the myosin molecule remain elusive. To date, there are no therapies targeted toward treating the underlying disease process of these inherited cardiomyopathies, due to lack of knowledge of how the mutations lead to hypertrophy. Technical challenges have been faced in studying the disease using animal models due to differences in background composition of myosin isoforms between species, and previous work using non- human myosin has produced mixed results. Recently, the production of functional human myosin in vitro has been established by using a mammalian cell line system, and we now have the capacity to obtain pure human cardiac myosin for functional analysis at the single molecule level. As a working hypothesis, we propose that mutations that lead to increased power output leading to HCM, and observed differences in clinical phenotype is related to degree of changes in force production. We hypothesize that these mutations affect the underlying power output capability of human ?-MyHC in different ways (e.g. changing the spring constant of the elastic element of the motor, changing duty ratio of the myosin) and that the eventual clinical phenotypes of HCM is a result of a particular fundamental mechanistic change in the human cardiac ?-MyHC. We further hypothesize that the clinical outcome in patients with HCM is directly related in the degree of changes in force production. To test these hypotheses, we test the following aims: Aim 1. Determine the fundamental biomechanical parameters (velocities at various loads, duty ratios, stroke sizes, and intrinsic force they produce) of HCM mutant human ?-cardiac myosins with mutations in the converter domain (R719W, R723G and G741R) that result in a malignant clinical phenotype. Aim 2. Compare the biomechanical parameters of HCM mutant human ?-cardiac myosins resulting in a malignant phenotype studied in Aim 1 with mutations at the same amino acid position resulting in benign phenotypes (R719W vs R719Q, R723G vs R723C and G741R vs G741W). Velocities and duty ratios will be measured using in vitro motility assays with purified actin, myosin and ATP. Stroke sizes and intrinsic force producing capability of single ?-cardiac myosin molecules will be measured using laser trap single molecule analysis. This study will be the first study to clarify the primary effect of mutation in human cardiac myosin for these mutations. It is an important study in connecting molecular genetics of a common cardiovascular disease and its phenotype at single molecule level, which will make significant impact on clinical management.